Mechanism of interaction between nuclease Dna2 and single-stranded DNA-binding protein RPA during DNA replication and repair

Single-stranded DNA as an intermediate widely exists in various DNA metabolism pathways
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In eukaryotes, the single-stranded DNA - binding protein RPA acts as a guardian of single-stranded DNA , and its presence is indispensable for both DNA replication and DNA repair .
RPA can not only protect the integrity of single-stranded DNA , but also guide downstream factors to signal or repair single-stranded DNA intermediates .
However, it is still unclear how the downstream enzymes and factors can outcompete RPA and bind to single-stranded DNA .
Among the many factors that interact with RPA , Dna2 is an evolutionarily conserved nuclease and plays a unique role in the digestion of RPA -bound single-stranded DNA , making Dna2 important for DNA replication following strand maturation as well as DNA It plays an important role in the repair of double-strand breaks .
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In DNA replication, the 5'-flap formed during lagging strand replication must be removed during lagging strand maturation
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In this process, Dna2 is involved in removing the long 5'-flap bound to RPA , allowing Fen1 nuclease to further remove the 5'-flap and promote lagging strand maturation .
During homologous recombination-mediated double-strand break repair,Dna2 cooperates with Sgs1 helicase to excise a segment of 5' - ssDNA at the end of the broken DNA to form 3' - ssDNA required for downstream template search and strand exchange .

On January 18 , 2022 , Qi Zhi's laboratory of Peking University Center for Quantitative Biology, Niu Hengyao 's laboratory of Indiana University, and Grzegorz Ira 's laboratory of Baylor College of Medicine published in the journal Nature Communications the title Deciphering the mechanism of processive ssDNA digestion by the Dna2 - Article on RPA ensemble .

The study found that in the eukaryotic model organism Saccharomyces cerevisiae, Dna2 can form a stable complex withRPA to " walk " on single-stranded DNA and processively digest single-stranded DNA (Figure 1 ), and the key to supporting this processive digestion is The cis- and trans-interfaces formed by the interaction of Dna2 and RPA .

Figure 1.

This study found that Dna2 can selectively form a stable ternary complex with RPA on a 5′- single-stranded DNA substrate (Figure 2 ), and revealed that the N -terminus of the Rfa1 large subunit in RPA is important for this ternary complex The formation of Dna2 is crucial, thus mechanistically explaining the previously reported selective activity of Dna2 towards 5' - ssDNA .

Figure 2.

The authors delved further and found that the NAB domain of the Rfa1 large subunit of RPA ( Rfa1-NAB ) can not only form a ternary complex with Dna2 on single-stranded DNA , but also support the processive digestion of Dna2 in place of RPA (Figure 3 ) .

Figure 3.

In the process of studying the fusion protein NAB-dna2- ∆ 248N , the authors found that although it can digest single-stranded DNA , it cannot effectively digest the complex secondary structure region contained in single-stranded DNA, and can not digest single-stranded DNA efficiently.

Figure 4.

Combining the results reported in this paper, the authors propose a novel model for the digestion of single-stranded DNA by the Dna2-RPA complex (Figure 5 )

Figure 5.

Shen Jiangchuan, a postdoctoral fellow at Indiana University, Dr.